The present invention relates to pharmaceutical compositions, methods, kits, cells and particles containing membranes derived from cells. More specifically, the invention relates to a novel method of altering the properties of cells and of particles with membranes derived from cells.
In 1993 Laukanen et al. have described a method that facilitates the addition of a hydrophobic membrane anchor to single chain variable fragment (scFv) antibody fragments expressed in E. coli (Laukanen et al., Protein Engineering 6:449-454,1993). This method relies on the fusion of the signal peptide and 9 N-terminal residues of the bacterial lipoprotein (lpp) to a scFv antibody fragment. It was previously shown by Ghrayeb et al (1984) that only the signal peptide and nine amino acids of the mature lpp are required for the correct processing and transport of an lpp fusion protein to the outer membrane of E. coli. Laukanen (Laukanen et al., Biochemistry 33: 11G64-11670,1994) and de Kruif (de Kruif et al., FEBS letters 399:232-236, 1996) demonstrated that the lipid-modified antibody fragment can be expressed in E. coli and that the lipid-modified scFv is inserted into he periplasmic leaflet of the outer membrane and not on the outside of tee bacterium. After preparation of membrane extracts from the E. coli cells and purification of the lipid-modified scFv, it was shown that these molecules retain their binding specificity and can be functionally reconstituted into artificial vesicles composed of a lipid bilayer.
Although possible, the loading of artificial vesicles requires a lengthy biochemical procedure which is not very efficient and requires large amounts of lipid-modified proteinaceous molecules for effective incorporation of said molecules into the artificial lipid bilayer. Moreover, the process is not very controllable as to the orientation of said molecules in the lipid bilayer. In addition, it was found that the resulting vesicles shed the lipid-modified proteinaceous molecules quite rapidly in vivo. These disadvantages are not easily bypassed by optimising the procedure since the underlying reasons for these phenomena are unknown. The limitations of the procedure severely inhibit the practical applications for artificial lipid bilayers loaded with lipid-modified proteinaceous molecules.
The most common approach to change the properties of cells is the introduction of DNA or RNA into the cells. Subsequent expression of the introduced nucleic acid leads to the presence of novel proteins in intracellular compartments or on the plasma membrane of the cells. DNA is introduced into cells by one of a variety of methods, where upon it is stably integrated in the genomic DNA or remains in the cells as an extra-chromosomal fragment. This strategy is widely used to study the function of the molecule encoded by the introduced DNA or to endow the recipient cell with properties encoded by the introduced DNA.
Cell therapy is a strategy for the treatment of many diseases. The aim of cell therapy is to replace or repair damaged tissue or organs or to enhance the biological function of cells. ?or cell therapy, cells are isolated from a tissue or organ and manipulated ex vivo, for example by adding growth factors to cells in tissue culture with the aim to increase their number (Gage et al, Nature 392 (supplement):18-24, 1998). It is also common practise in cell therapy to introduce DNA into cells and, by using selectable markers such as antibiotic resistance, select cells that stably or transiently express the introduced DNA. The aim of this approach is to endow the recipient cell with novel properties. For example, DNA encoding a growth factor receptor may be introduced ex vivo into bone marrow cells and cells that express the growth factor receptor may be re-infused into a recipient organism. Treatment of the organism with the growth factor then results in the in vivo expansion of manipulated bone marrow cells expressing the growth factor receptor (Jin et al, Proc. Natl. Acad. Sci. USA 95:8092-8097,1998). In another application, T lymphocytes may be retrovirally transduced with DNA encoding a fusion protein consisting of a scFv antibody specific for folate-binding protein joined to the Fc-receptor gamma chain. After selection and expansion of T cells expressing the fusion protein and re-infusion, it was shown in an animal model that the T lymphocytes reacted against tumour cells (Hwu et al., Cancer Res. 55:3369-3373, 1995). The method of introducing DNA into cells with the aim to alter their properties is time-consuming, requires the introduction of foreign genetic material into human cells and is constrained by the inefficiency of DNA transfer into some cell types, especially freshly-isolated cells.
The present invention discloses a novel use of lipid-modified proteinaceous molecules. In the present invention lipid-modified proteinaceous molecules are contacted with cells and/or with particles containing membranes derived from cells. Upon contact the lipid-modified proteinaceous molecules are inserted into the membrane of a cell and/or of a particle with a membrane derived from a cell. This novel use not only enables a completely novel approach to manipulate cells and/or particles containing membranes derived from cells. But in addition, this process is very efficient, requiring relatively low amounts of lipid-modified proteinaceous molecules for effective incorporation by a cell and or a particle derived from a cell. Moreover, the lipid-modified proteinaceous molecules added to said cell and/or said particle are surprisingly stable in vivo. The proteinaceous molecules to be taken up by said cells and/or said particles are produced as lipid-modified molecules that spontaneously attach to membranes without using biochemical coupling procedures. This procedure can be used to endow the recipient cells with molecules that act as signaling molecules or otherwise affect the properties of the cells, for example in their capacity to recognise a target or to home to a particular tissue or organ. The present invention provides a rapid and effective alternative for methods that rely on introduction and expression of nucleic acids into cells, resulting in the expression of plasma membrane-associated proteinaceous molecules. Since the present invention is independent of gene transfer and rapid the invention possesses discrete advantages over the conventional methods based on gene transfer. Although many gene transfer methods have been devised some cell types have remained refractory to efficient gene transfer. The present invention is independent of gene transfer efficiency and thus provides a completely different and versatile method for the insertion of proteinaceous molecules in the membranes of cells and/or of particles with membranes derived from cells. Moreover, the process of adding the proteinaceous molecules is fast, thus obviating the obligatory incubation period for obtaining maximal protein expression following gene transfer.
One of the most prominent applications of the present invention lies in the field of cell therapy as a strategy for the treatment of human diseases.
The present invention provides a novel method to alter the properties of cells and/or of particles containing membranes derived from cells. The present invention does not rely on the transfer of a nucleic acid but instead directly supplies the desired protein to the membrane enabling a rapid alteration of the properties of the cell and/or the particle with a membrane derived from a cell. The present invention offers advant ages over conventional nucleic acid transfer in that it is fast and suitable for a wide variety of cell types including but not limited to primary cells isolated from a human.
One aspect of the invention is to provide a method for efficiently attaching proteinaceous molecules to the membranes of cells and or of particles containing membranes derived from cells. The method is rapid, leads to the incorporation of high numbers of molecules in the outer membrane, requires minimal handling of said cells and/or said particles, does not require introduction of genetic material into cells and appears to be generally applicable to all cell types and all particles containing membranes derived from cells. A major advantage for therapeutical application is that the method can be used to efficiently incorporate proteinaceous molecules in the plasma membrane of cells that are freshly-isolated from a tissue or organ, which difficult to achieve with other methods. In addition, the method offers the advantage that attachment of molecules to the plasma membrane of cells can be accomplished in a very short period of time.
In one embodiment of the invention a process is provided for providing a cell and/or a particle with a membrane derived from said cell with an additional proteinaceous molecule wherein said process comprises contacting said cell or said particle with a proteinaceous molecule, said molecule comprising at least one hydrophobic moiety. The method of contacting may be any method wherein the lipid-modified proteinaceous molecule is presented from the outside to said cell and/or said particle. In one embodiment of the invention said hydrophobic moiety comprises a stretch of hydrophobic amino acids capable of inserting itself in said membranes. In another embodiment of the invention said hydrophobic part comprises one or more fatty acid chains. In a preferred embodiment, the invention provides a process for providing a cell and/or a particle with a membrane derived from a cell with an additional proteinaceous molecule said process comprising contacting said cell and/or said particle with a lipid-modified proteinaceous molecule. In this preferred embodiment the proteinaceous molecule is linked to one or more lipids, a process termed lipidation, as a result of one or more lipidation signals. One lipidation signal resulting in one lipid tail is sufficient for a rapid transfer and anchorage of the lipid-modified proteinaceous molecule to the outer membrane of a cell and/or of a particle with a membrane derived from a cell. However, a second or even more lipidation signals, thus resulting in a lipid-modified proteinaceous molecule with two or more lipid tails, may be added for increased stability of the molecule in the membrane or to increase the stability of a specifically desired three dimensional configuration of said molecule. For example, the construct depicted in FIG. 1 may be modified to contain a second lipidation signal at the 3 prime end of the scFv gene, resulting in a lipid-modified scFv with a lipid tail at both the amino and the carboxyl terminus of the protein.
In one embodiment of the invention the lipidation of the proteinaceous molecule may occur in a cell free system where the lipidation as a result of the lipidation signal is achieved by components added to the cell free system (see for instance Rusinol, A. E. J et al. Biol. Chem. 272, 8019-8025, 1997). In a preferred embodiment of the invention, the lipidation of the proteinaceous molecule is accomplished in a cell. In this preferred embodiment of the invention cellular enzymes are recruited to catalyse the lipidation of the proteinaceous molecules following a signal that is recognised by the lipidation machinery of the cell. In a particularly preferred embodiment of the invention the lipidation of the proteinaceous molecules is performed in bacteria in response to a lipidation signal recognised by the bacterial lipidation machinery. Production of a lipid-modified proteinaceous molecule in bacteria compared to eukaryotic cells generally results in higher yields. Production in bacteria is more cost effective than production in eukaryotic cells. Production of lipid-modified proteinaceous molecules in bacteria, as opposed to eukaryotic cells, for a pharmaceutical application in human and/or animal has furthermore the advantage that bacterial produced pharmaca have a significantly lower propensity for the presence of viruses and/or prions that may be harmful for a human and/or an animal. In one aspect of this particularly preferred embodiment the limidation of the proteinaceous molecules occurs in E. coli and the lipidation signal is derived from bacterial lipoprotein. In this particularly preferred embodiment the synthesis and the lipidation of said proteinaceous molecule is accomplished by introducing a recombinant DNA expression plasmid or vector into E. coli. Glycosylphosphatidylinositol (GPI)-linked proteins form another non-limiting example of a group of proteins from of which the lipidation signal may be incorporated into a proteinaceous moiety to produce the lipid-modified proteinacecus molecules of the invention. GPI-linked proteins are plasma membrane molecules that lack a cytoplasmic tail and are attached to the plasma membrane of cells by a lipid anchor. Despite the lack of a cytoplasmic tail, GPI-linked proteins may operate as signaling molecules, conveying signals to the cell after binding of ligards or antibodies. Cell signaling via GPI-linked proteins may induce a broad variety of cellular responses, including cell activation and differentiation, apoltosis, and secretion of cytokines. Available evidence suggests that signaling via GPI-linked proteins may occur through the physical interaction of the GPI-linked protein with other membrane molecules (Simons et al., Nature 387;569-572, 1997).
In a preferred embodiment of the invention proteinaceous molecules are lipidated as a result of a lipidation signal derived from glycosylphosphatidylinositol (GPI)-linked proteins. In this preferred embodiment the lipidation of the proteinaceous molecules is achieved in eukaryotic cells, preferably yeast cells. Sequences containing the signal leading to the attachment of glycosylphosphatidylinositols moieties to proteins may be found in Udenfriend et al, Annu. Rev. Biochem. 64, 563-591 1995.
Many different paoteinaceous molecules may be used in the present invention. Proteinaceous molecules may be derived from proteins present in nature but may also be generated completely artificially as long as they contain or have added to them a lipidation signal. In a preferred embodiment of the invention the proteinaceous moiety of the lipid-modified proteinaceous molecules is derived from natural proteinaceous molecules with actions near or in membranes. Non-limiting examples of such molecules are receptors, co-receptors, (membrane-bound) ligands, signaling molecules, homing molecules or molecular pumps. on the other hand however, also molecules may be used with no known action in membranes or with actions that normally do not depend on the presence of a membrane.
Artificial proteinaceous molecules, e.g. not present in nature, can upon lipidation also be used for the present invention. Non-limiting examples of this are lipid-modified single chain variable fragments (scFv). Applications of such lipid-modified scFv are manifold, for instance but not limited to application of lipid-modifed scFv specific for a certain type of cell. Such molecules are useful to target cells of the immune system to specific cells in the body thus interfering either positively or negatively with the immune system. One application is to enable a more effective immune response against undesired cells such as malignant cells or virus infected cells. Another application is to interfere with the immune system in a negative way to suppress an undesired immune response and induce a specific tolerance such as is desired in the most common forms of arthritis, insulin-related diabetes or allergies. In these diseases part of the immune system is inadvertently directed to self-antigens or over sensitive to foreign antigens.
In another aspect of the invention the proteinaceous molecule is derived from a proteinaceous molecule active in the immune system so a human or animal. Non-limiting examples are antibodies, fragments derived from antibodies such as fragment antigen binding (FAB) fragments, proteins resembling fragments derived from antibodies such as artificially produced FAB-fragments and T-cell receptors.
In addition, the proteinaceous molecule may be a derivative from other classes of proteins derived from cells of the immune system such as co-stimulatory molecules, heat shock proteins, major histo-compatibility complex (MHC) proteins or antigenic peptides. FAB-fragments generated by cleavage from antibodies or FAB-fragment-like proteins generated artificially will further collectively be referred to as FAB-fragments, In another aspect of the invention a lipid-modified proteinaceous molecule comprises a stretch of amino acids conferring to the proteinaceous molecule the property to interact with a signal-transducing molecule present on the plasma membrane of a cell.
In one aspect of the invention a cell, or a particle with a membrane derived from a cell, is contacted with two or more different types of lipid-modified proteinaceous molecules. The crucial difference being the capability of the lipid-modified proteinaceous molecules to change the property of said cell or said particle in a different way. In this aspect of the invention two or more different types of lipid-modified proteinaceous molecules are used to combine the effect of each type of lipid-modified proteinaceous molecule.
In yet another aspect of the invention the lipid-modified proteinaceous molecules contain an additional signal, designated xe2x80x9cpurificationxe2x80x9d tag enabling the easy purification of the lipid-modified proteinaceous molecules during the production process. A non-limiting example of such a purification tag is a polyhistidine sequence or polyhistidine tag. In another embodiment of the invention the lipid-modified proteinaceous molecules contain an additional signal, designated xe2x80x9cdetectionxe2x80x9d tag for the detection of the lipid-modified proteinaceous molecules. A non-limiting example of such a detection tag is a short stretch of amino acids derived from the myc-gene product, a so-called myc tag.
In another aspect of the invention is provided a kit with which the invention can be practised to obtain a cell or a particle with a membrane derived from said cell, comprising an additional lipid-modified proteinaceous molecule. This kit minimally contains a lipid-modified proteinaceous molecule but may further contain matters and substances useful to operate the invention such as sterile bags, culture materials, buffers and quality assurance materials such as materials in the form of scFv to assay the amount of lipid-modified proteinaceous molecule loaded onto a cell or a particle containing a membrane derived from a cell.
In one embodiment of the invention is provided a vector for the production of lipid-modified proteinaceous molecules in cell, preferably bacterial or yeast cells. Said vector comprises at least one open reading frame which minimally encodes for at least one protein of interest and at least one lipidation signal. Said open reading frame may further comprise additional elements coding for a detection tag and/or a purification tag.
It is clear to a person skilled in the art that only the essential part or parts of a protein are required in the lipid-modified proteinaceous molecules of the invention. Thus deletions/insertions or mutations in non-relevant parts of the protein molecule of which the proteinaceous moiety in the lipid-modified proteinaceous molecule is derived are anticipated to be equally effective as the entire protein molecule.
It is also clear to a person skilled in the art that the protein moiety of the lipid-modified proteinaceous molecule may contain further functional units derived from, different proteins existing in nature or artificial to broaden the functionality of said lipid-modified proteinaceous molecule.
It is clear that the lipid-modified proteinaceous molecules of the invention, when contacted with a cell or a particle with a membrane derived from a cell, will preferentially attach to first membrane encountered and orient themselves such that the proteinaceous moiety is directed outward. However, active processes, such as endocytosis, lead to entry of the lipid-modified proteinaceous molecules or derivatives thereof into the interior of the cell or the particle with a membrane derived from a cell.
An important aspect of the invention is the alteration of the properties of a cell or a particle containing a membrane derived from a cell by providing said cell or said particle with additional lipid-modified proteinaceous molecules. Said cell may be any type of prokaryotic or eukaryotic cell. In a preferred embodiment of the invention said cell is a human cell. In a preferred embodiment of the invention the properties of tumour infiltrating lymphocytes (TILs) or lymphocyte activated killer cells (LAKs) are altered by providing the cells with additional lipid-modified proteinaceous molecules. TILs and LAKs are cells of the immune system that have been expanded in vitro with interleukin-2 (IL-2) to obtain large numbers of cells that have anti-tumour effect. After in vitro expansion, these cells are re-infused into patients with cancer where they can exert their anti-tumour effect (Rosenberg et al, N. Eng. 4. Med. 319, 1676-1680, 1988; Rosenberg et al, Clin. Oncol. 10, 180-199, 1992). Infusion of polyclonal populations of TILs has yielded low response rates, short response duration""s, poor localisation of the T-cells to tumour sites and severe toxicity""s associated with concurrent administration of high doses of IL-2 (Yee et al, Current Opin. Immunol. 9, 702-708, 1997). To overcome some of these problems, in vivo expanded T-cells have been transfected with DNA encoding receptors for recognition of tumour cells. In some applications, DNA encoding a scFv has beer. introduced in in vitro expanded T-cells (Eshar et al, Proc. Natl. Acad. Sci. USA 90, 720-724,1993; Brocker et al, Eur. J. Immunol. 23, 1435, 1993). In vivo studies with such gene-modified T-cells, endowed with a scFv specific for tumour cells have shown promising results (Abken et al, Immunol. Today, 19, 2-5, 1998). A drawback of such approaches is the use of gene-modified cells for therapy and the efficiency of producing sufficient numbers of gene-modified cells (Abken et al, Cancer Treat. Rev. 23 (2), 97-112, 1997; Abken et al, Immunol. Today, 19, 2-5, 1990). By using lipid-modified scFv, these limitations can be overcome. This approach does not involve introduction of genes into cells to be re-infused in patient and is the addition of the desired scFv to the cells is very efficient.
Thus in a preferred embodiment of the invention TILs or LAKs are supplied with additional lipid-modified proteinaceous molecules, preferably lipid-modified scFv, to alter the properties of these cells, preferably a property enabling or improving the homing of said TILs or LAKs to tumour sites. In a preferred embodiment of this invention said lipid-modified scFv are directed to endothelial cells of growing blood vessels, preferably directed to blood vessels in tumours.
In a preferred embodiment of the invention particles with membranes derived from cells are contacted with lipid-modified proteinaceous molecules. Particles containing membranes derived from cells are particles that are for example formed after mechanical disruption of cells or solubilisation of cells in detergents. Particles containing membranes derived from cells are also produced by living cells such as for example exosomes (Escola et al., J. Biol. Chem. 273, 20121-20127,1998 and Zitvogel et al., Nat. Med. 4, 594-600, 1998) or such as enveloped viruses, very low density lipoprotein (VLDL), low density lipoprotein (LDL) and chilomicron particles. Particles with membranes derived from cells may be provided with additional lipid-modified proteinaceous molecules to alter the properties of said particles. Lipid-modified proteinaceous molecules may be contacted with said particles directly or alternatively, lipid-modified proteinaceous molecules may be contacted with cells producing said particles. In the latter case said lipid-modified proteinaceous molecules are incorporated into said particles during their production. In a preferred embodiment of the invention said lipid-modified proteinaceous molecules provide the particles with membranes derived from cells with a new target cell specificity. Such specificity may be added through, for example, a scFv molecule with a specificity for an antigen on the surface of a cell type previously not belonging to the target cell pool of said particle.
The present invention provides methods for the addition of lipid-modified proteinaceous molecules to the membranes of cells and/or of particles with membranes derived from cells.
The present invention is useful to solve one of the problems associated with viral vectors used in the transfer of foreign genetic to target cells. In therapeutic applications of viral vectors in the field of gene therapy, target cells consists of cells from an entire organ such as the liver or an entire group of cells dispersed in the body such as the hematopoietic stem cells, metastasised tumour cells or virus infected cells. Is it for delivery of foreign genetic material to organs sometimes possible to deliver the viral vector only to the cells of that organ (for instance through physical means or surgery). For dispersed target cells, delivery of the viral vector to the target cells can only be accomplished through the bloodstream. To avoid uptake of the viral vector by other cells, some kind of specificity for the target cell must be introduced into the viral vector. This so-called targeting of viral vectors is a very active field of research. For enveloped viruses, i.e. surrounded by a membrane, most approaches to targeting of viral vectors depend on modification of the viral envelop protein. Said viral envelop protein is present on the outside of the viral membrane and is responsible for target cell recognition and, in most cases, for fusion of the viral membrane with a cellular membrane. Approaches to modify the viral envelop protein and other targeting approaches such as those that rely on the incorporation of specific viral (co)-receptors in the membrane of the virus particle have the serious disadvantage that changing the targeting specificity is a lengthy and rather unpredictable process. In addition, more often than not the viral vector provided with the new target cell specificity is much less efficiently produced, or much less infective when compared to the unmodified virus. With the methods of the invention, enveloped viral vectors may be provided with novel target cell specificities in a rapid, reproducible way.
In a preferred embodiment of the invention a cell is genetically modified before or after being contacted with lipid-modified proteinaceous molecules. For example, it is advantageous to introduce a gene encoding the Herpes Simplex Virus (HSC) thymidine kinase (tk) into the cell as a build in safeguard against undesired effects of said cells once inside the body. The cells expressing the HSV-tk are sensitive to killing by the drug gangcyclovir (Freeman et al, SM Lancet 349, 2-3, 1997). Another example is the introduction of a cytokine gene such as IL-2 into a tumour cell treated with lipid-modified proteinaceous molecules, wherein expression of IL-2 enhances the immune response against the tumour cells. We have developed a procedure that allows the rapid and efficient insertion of lipid-tagged (LT) proteins into the membrane of prokaryotic and eukaryotic cells. The procedure appears to be applicable to most eukaryotic cell types including freshly isolated cell populations. LT-scFv antibody fragments inserted into cell membranes were shown to remain fully functional, capable of binding soluble and membrane-bound molecules expressed by other cells. LT-scFv specific for molecules expressed by antigen presenting cells and attached to the membrane of tumor cells mediated efficient phagocytosis of the tumor cells. Because of the ease and efficiency of production and purification of LT-proteins and the method of attaching them to freshly isolated tumor cells, this approach may be used in cellular vaccination strategies with modified autologous tumor cells capable of eliciting a vigorous anti-tumor response.
His-tagged LT-scFv""s were expressed in E. coli and purified using metal affinity chromatography. To achieve even higher purity, additional affinity chromatography employing the myc tag fused to the LT-scFv fragment and an anti-myc monoclonal antibody may be used (Laukkanen el al. 1994, Biochemistry. 33:11664-11670). The N-terminal portion of the recombinant lipoprotein consists of a glyceryl cysteine to which three fatty acids are attached. This lipid group is highly hydrophobic and its natural function is to anchor bacterial LPP within the outer membrane of E. coli (Grayeb et al. 1984, J. Biol. Chem. 259:463-467). We presume that the lipid-modified proteins localize in the cell membranes in a similar fashion.
Peptides and proteins, including scFv antibody fragments, have been displayed on the surface of a variety of cell types including filamentous phage particles (Smiths, G. P. 1985, Science. 228:1315-1317), bacteria (Francisco, J. A. et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2713-2717), yeast Boder E. T. et al. 1997, Nat. Biotechnol. 15:553-557) and mammalian cells (Eshhar, Z. 1997, Cancer Immunol. Immunother. 45:131-136). In these systems, surface display is achieved by genetic fusion of the DNA encoding the protein of interest to DNA encoding a host cell surface or coat protein, followed by introduction of the fusion construct into the host cell and selection of transfected cells. The method described here differs from these approaches in that display of lipid-modified proteins by membrane insertion does not require introduction of DNA into the cell nor does it require culture and selection of cells. Membrane-insertion of lipid-modified proteins is completed within 30 minutes, requires a simple incubation and washing step and is nearly 100% efficient. CD14 and the Fcxcex3 receptors CD32 and CD64 belong to a group of cell surface receptors engaged in phagocytosis by various types of phagocytes including macrophages, dendritic cells, and monocytes (Kwiatkowska, K. et al. 1999, BioEssays. 21:422-431). CD14 mediates the recognition and phagocytosis of apoptotic cells via an as yet unidentified ligand expressed by apoptotic but not by viable cells (Devitt, A. et al. 1998, Nature 392:505-509), whereas CD32 and CD64 mediate phagocytosis of IgG-opsonized particles such as microorganisms. We reasoned that an anti-CD14 LT-scFv fragment anchored to the cell membrane of a viable cell could be used to mimic the putative CD14 ligand on apoptotic cells.
Similarly, we anticipated that LT-scFv""s specific for CD32 and CD64 would render the recipient cell susceptible to phagocytosis mediated via Fcxcex3 receptors. Indeed, cells modified with LT-scFv specific for CD14, CD32 and CD64 were efficiently phagocytosed by monocytes and macrophages. We conclude that LT-scFv anchored to cell membranes act as surrogate receptors that are capable of altering the properties of the recipient cell.
Monocytes, macrophages and dendritic cells also function as antigen presenting cells. Uptake of antigen-antibody complexes via Fcxcex3 receptors such as CD32 and CD64 results in the capture and internalization of low concentrations of antigens, followed by processing and presentation to T lymphocytes For example, when antigens are internalized as immune complexes through CD32 by dendritic cells there is a marked increase in the efficiency of T cell activation in response to low concentrations of antigen (Sallusto, F et al. 1994, J. Exp. Med. 179: 1109-1114). Targeting antigen to either CD32 or CD64 on monocytes reduces the concentration of antigen required for T cell activation approximately 1000-fold (Gosselin, E. J. et al. 1932, J. Immunol. 149:3477-3481). Peptides fused to anti-CD64 mAb 22, used as an LT-scFv fragment in this study, were recently shown to be up to 1000-fold more efficient than the non-fused peptides for T cell stimulation when targeted to monocytes (Liu, C. et al. 1999, J. Clin. Invest. 98:2001-2007). Extrapolating from these data, we predict that targeting irradiated tumor cells to antigen-presenting cells via membrane bound LT-scFv""s will be an attractive method for activation of anti-tumor T cell responses. Notably, the method does not require identification and cloning of tumor antigens but instead uses the whole tumor cell as a source of tumor antigens.
Furthermore, more than one specificity of scFv may be displayed on a single tumor cell. Thus, incorporation of an anti-CD40 LT-scFv, in addition to a targeting antibody for antigen presenting cells, may replace the requirement for TH cells in the activation of antigen presenting cells by mimicking the CD40 ligand (Ridge, J. P. et al. 1998, Nature. 393:474-478) Ex vivo alteration of the properties of autologous tumor cells and re-infusion of the modified cells in cancer patients, or animal models of cancer, has been actively explored as a vaccination strategy for the treatment of cancer. Collectively, these approaches aim at increasing the immunogenicity of the tumor cells resulting in the induction of anti-tumor responses. Tumor cells have been genetically modified to secrete cytokines or express co-stimulatory molecules that stimulate cells of the immune system (Nawrocki, S. et al. 1999, Cancer Treat. Rev. 25:29-46). Alternatively, tumor cells have been modified by viral infection (Schirrmacher, V., et al. 1999, Gene Ther. 6:63-73), bispecific molecules (Haas, C. et al. 1999, Cancer Gene Ther. 6:254-262), haptenization of membrane molecules (Berd, D. et al. 1998, Semin. Oncol. 25: 646-653)or by fusion of tumor cells and antigen presenting cells to generate hybrids with characteristics of both the tumor and antigen presenting cell (Gong, J. et al. 1997, Nat. Med. 3:558-561). Non-modified, irradiated tumor cells have been injected into cancer patients in combination with adjuvants with considerable clinical effect (Vermorken, J. B. et al. 1999, Lancet 353:345-350). Insertion of lipid-modified proteins in the cell membrane of cancer cells builds on these findings and provides a vaccination strategy with autologous tumor cells with tailor-made properties. For example, it is within the scope of the invention to irradiated autologous tumor cells, endowed with LT-scFv specific for antigen-presenting cells and cytokines that stimulate antigen presenting cell expansion and maturation, are able to induce a vigorous anti-tumor response. Lipid-modification and expression of proteinaceous molecules in E. coli and their insertion into cell membranes is a general approach to temporarily endow a cell with single or multiple novel properties.